Manipulation of Cells on a Droplet Actuator

ABSTRACT

A method of inoculating a culture medium including providing a droplet including a single cell type on a droplet actuator and inoculating a culture medium with the droplet. A method of providing a metabolically useful substance to a cell culture, including providing a droplet actuator including a cell culture droplet loaded thereon, the sample droplet including cells and a cell culture medium, and a second droplet comprising a metabolically useful substance. The method also includes conducting one or more droplet operations to combine the cell culture droplet with the second droplet on the droplet actuator. Related methods, droplet actuators, and systems are also provided.

RELATED APPLICATIONS

This application claims priority to U.S. Patent Application Nos.61/013,535, entitled “Manipulating cells in a droplet actuator,” filedon Dec. 13, 2007; and 61/091,637, entitled “Manipulating cells in adroplet actuator,” filed on Aug. 25, 2008; the entire disclosures ofwhich are incorporated herein by reference.

GRANT INFORMATION

This invention was made with government support under DK066956-02awarded by the National Institutes of Health of the United States. TheUnited States Government has certain rights in the invention.

FIELD OF THE INVENTION

The inventions relates to methods, devices and systems for sortingcells, inoculating culture media, replenishing culture media, growingcells, and testing cell cultures.

BACKGROUND OF THE INVENTION

Droplet actuators are used to conduct a wide variety of dropletoperations, such as droplet transport and droplet dispensing. A dropletactuator typically includes two surfaces separated by a gap. One or bothsurfaces include electrodes for conducting droplet operations. The gaptypically includes one or more filler fluids that are relativelyimmiscible with the droplets. Droplets may, for example, be reagentsand/or droplet fluids for conducting assays. In wide variety ofapplications, such as the production of antibodies and assaying stemcells, samples within droplet actuators may include cells to bemanipulated and, therefore, there is a need for new approaches tomanipulating cells within a droplet actuator.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides a method of inoculating a culture medium. Themethod may include providing a droplet including a single cell type on adroplet actuator and inoculating a culture medium with the droplet. Theinoculating step may involve conducting one or more droplet operationsto bring the droplet into contact with the culture medium. The dropletincluding a single cell type may be provided by (a) providing a dropletactuator including a sample droplet loaded thereon, the sample dropletincluding cells of multiple cell types; (b) dispensing a sub-dropletfrom the sample droplet; (c) analyzing the sub-droplet to determinewhether the sub-droplet includes a single cell type; (d) repeating steps(b) and (c) until one or more droplets each including a single cell or asingle cell type is identified.

The invention also provides a method of providing a droplet including asingle cell type, the method including: providing a droplet actuatorincluding: a sample droplet loaded thereon, the sample droplet includingcells of multiple cell types; a bead droplet including one or more beadshaving affinity for a specific one of the cell types; conducting one ormore droplet operations to combine the bead droplet with the sampledroplet, thereby permitting cells of the specific one of the cell typesto bind to the beads; conducting a droplet based washing protocol toseparate the beads bound to cells of the specific one of the cell typesfrom cells of other cell types.

Further, the invention provides a method of providing a droplet having amodified distribution of cell types, the method including: providing adroplet actuator including a sample droplet loaded thereon, the sampledroplet including a first distribution of cells of multiple cell types;activating a series of electrodes to elongate the droplet, therebyproviding an elongated droplet; applying a dielectrophoretic gradientalong the elongated droplet; deactivating an intermediate one of theseries of electrodes to divide the droplet into two or moresub-droplets, each such sub-droplet having a distribution of cells thatdiffers from the first distribution of cells of multiple cell types. Atleast one of the sub-droplets provided may include a cell type that isenriched relative to the sample droplet. The cell culture droplet andthe second droplet may be situated between droplet actuator substratesin proximity to a droplet operations surface. The cell culture dropletmay be substantially surrounded by a filler fluid.

In another embodiment, the invention provides a method of providing ametabolically useful substance to a cell culture. A droplet actuator maybe provided, including: and a cell culture droplet loaded thereon, thesample droplet including cells and a cell culture medium; a seconddroplet including a metabolically useful substance. The method mayinclude conducting one or more droplet operations to combine the cellculture droplet with the second droplet on the droplet actuator. Thecell culture droplet may be situated between droplet actuator substratesin proximity to a droplet operations surface. The cell culture dropletand the second droplet may be situated between droplet actuatorsubstrates in proximity to a droplet operations surface. The cellculture droplet may be substantially surrounded by a filler fluid. Thecell culture droplet and the second droplet may be substantiallysurrounded by a filler fluid.

The invention provides a method of growing cells on a droplet actuator.The method includes providing a droplet actuator including a cellculture droplet including a cell culture medium and one or more cells;and maintaining the droplet at a temperature suitable for causing thecells to grow in the cell culture medium on the droplet actuator. Thecell culture droplet may be situated between droplet actuator substratesin proximity to a droplet operations surface. The cell culture dropletmay be surrounded by a filler fluid. The cells may include cells boundto beads.

The invention also provides a method of providing a hybridoma. A dropletactuator may be provided including: a B-cell droplet including a B-cell;and a myeloma cell droplet including a myeloma cell. The method mayinvolve conducting droplet operations to combine the B-cell droplet withthe myeloma cell droplet under conditions suitable to cause the fusionof the B-cell with the myeloma cell to produce a hybridoma. The B-celldroplet is situated between droplet actuator substrates in proximity toa droplet operations surface. The myeloma cell droplet is situatedbetween droplet actuator substrates in proximity to a droplet operationssurface. The hybridoma may be grown and tested on the droplet actuator.The B-cell droplet may be surrounded by a filler fluid. The myeloma celldroplet may be surrounded by a filler fluid.

In a further embodiment, the invention provides a method of monitoring acell culture. The method may include providing droplet actuatorincluding a cell culture droplet including a cell culture medium and oneor more cells; conducting one or more droplet operations to dispense asample droplet from the cell culture medium; and testing the sampledroplet for one or more target substances. The cell culture droplet maybe situated between droplet actuator substrates in proximity to adroplet operations surface. The sample droplet may be dispensed usingelectrode-mediated droplet operations between droplet actuatorsubstrates in proximity to a droplet operations surface. Testing may beeffected using steps including electrode-mediated droplet operationsbetween droplet actuator substrates. The cell culture droplet may besubstantially surrounded by a filler fluid. The sample droplet may besubstantially surrounded by a filler fluid.

Testing may be effected while the sample droplet is substantiallysurrounded by a filler fluid. Testing may involve conducting one or moreelectrode-mediated, droplet-based assays on the droplet actuator. Thetarget substances may include metabolically useful substances.

One or more droplet operations may be used to replace the sample dropletfrom the cell culture droplet with a replacement droplet added to thecell culture droplet, the replacement droplet including one or moremetabolically useful substances. The replacement droplet is dispensedand transported from a droplet actuator reservoir by electrode mediateddroplet operations into contact with the cell culture droplet. Thereplacement droplet may be selected to replace one or more specificsubstances identified as deficient in the testing step. The testing andreplacement of one or more target substances may be automated. Thetesting may include quantifying one or more metabolic substances.

The invention also provides a method of monitoring a cell cultureincluding: providing cell culture including a cell culture medium andone or more cells and a fluid path to a droplet actuator; providing acell culture droplet from the cell culture to the droplet actuator viathe fluid path; testing the sample droplet for one or more metabolicallyuseful substances. The method may also include replacing one or moremetabolically useful substances identified as deficient by the testingstep.

Definitions

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting achange in the electrical state of the one or more electrodes which, inthe presence of a droplet, results in a droplet operation.

“Bead,” with respect to beads on a droplet actuator, means any bead orparticle that is capable of interacting with a droplet on or inproximity with a droplet actuator. Beads may be any of a wide variety ofshapes, such as spherical, generally spherical, egg shaped, disc shaped,cubical and other three dimensional shapes. The bead may, for example,be capable of being transported in a droplet on a droplet actuator orotherwise configured with respect to a droplet actuator in a mannerwhich permits a droplet on the droplet actuator to be brought intocontact with the bead, on the droplet actuator and/or off the dropletactuator. Beads may be manufactured using a wide variety of materials,including for example, resins, and polymers. The beads may be anysuitable size, including for example, microbeads, microparticles,nanobeads and nanoparticles. In some cases, beads are magneticallyresponsive; in other cases beads are not significantly magneticallyresponsive. For magnetically responsive beads, the magneticallyresponsive material may constitute substantially all of a bead or onecomponent only of a bead. The remainder of the bead may include, amongother things, polymeric material, coatings, and moieties which permitattachment of an assay reagent. Examples of suitable magneticallyresponsive beads are described in U.S. Patent Publication No.2005-0260686, entitled, “Multiplex flow assays preferably with magneticparticles as solid phase,” published on Nov. 24, 2005, the entiredisclosure of which is incorporated herein by reference for its teachingconcerning magnetically responsive materials and beads. The fluids mayinclude one or more magnetically responsive and/or non-magneticallyresponsive beads. Examples of droplet actuator techniques forimmobilizing magnetically responsive beads and/or non-magneticallyresponsive beads and/or conducting droplet operations protocols usingbeads are described in U.S. patent application Ser. No. 11/639,566,entitled “Droplet-Based Particle Sorting,” filed on Dec. 15, 2006; U.S.Patent Application No. 61/039,183, entitled “Multiplexing Bead Detectionin a Single Droplet,” filed on Mar. 25, 2008; U.S. Patent ApplicationNo. 61/047,789, entitled “Droplet Actuator Devices and DropletOperations Using Beads,” filed on Apr. 25, 2008; U.S. Patent ApplicationNo. 61/086,183, entitled “Droplet Actuator Devices and Methods forManipulating Beads,” filed on Aug. 5, 2008; International PatentApplication No. PCT/US2008/053545, entitled “Droplet Actuator Devicesand Methods Employing Magnetic Beads,” filed on Feb. 11, 2008;International Patent Application No. PCT/US2008/058018, entitled“Bead-based Multiplexed Analytical Methods and Instrumentation,” filedon Mar. 24, 2008; International Patent Application No.PCT/US2008/058047, “Bead Sorting on a Droplet Actuator,” filed on Mar.23, 2008; and International Patent Application No. PCT/US2006/047486,entitled “Droplet-based Biochemistry,” filed on Dec. 11, 2006; theentire disclosures of which are incorporated herein by reference.

“Droplet” means a volume of liquid on a droplet actuator that is atleast partially bounded by filler fluid. For example, a droplet may becompletely surrounded by filler fluid or may be bounded by filler fluidand one or more surfaces of the droplet actuator. Droplets may, forexample, be aqueous or non-aqueous or may be mixtures or emulsionsincluding aqueous and non-aqueous components. Droplets may take a widevariety of shapes; nonlimiting examples include generally disc shaped,slug shaped, truncated sphere, ellipsoid, spherical, partiallycompressed sphere, hemispherical, ovoid, cylindrical, and various shapesformed during droplet operations, such as merging or splitting or formedas a result of contact of such shapes with one or more surfaces of adroplet actuator. For examples of droplet fluids that may be subjectedto droplet operations using the approach of the invention, seeInternational Patent Application No. PCT/US 06/47486, entitled,“Droplet-Based Biochemistry,” filed on Dec. 11, 2006. In variousembodiments, a droplet may include a biological sample, such as wholeblood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum,cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion,serous fluid, synovial fluid, pericardial fluid, peritoneal fluid,pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastricfluid, intestinal fluid, fecal samples, liquids containing single ormultiple cells, liquids containing organelles, fluidized tissues,fluidized organisms, liquids containing multi-celled organisms,biological swabs and biological washes. Moreover, a droplet may includea reagent, such as water, deionized water, saline solutions, acidicsolutions, basic solutions, detergent solutions and/or buffers. Otherexamples of droplet contents include reagents, such as a reagent for abiochemical protocol, such as a nucleic acid amplification protocol, anaffinity-based assay protocol, an enzymatic assay protocol, a sequencingprotocol, and/or a protocol for analyses of biological fluids.

“Droplet Actuator” means a device for manipulating droplets. Forexamples of droplet actuators, see U.S. Pat. No. 6,911,132, entitled“Apparatus for Manipulating Droplets by Electrowetting-BasedTechniques,” issued on Jun. 28, 2005 to Pamula et al.; U.S. patentapplication Ser. No. 11/343,284, entitled “Apparatuses and Methods forManipulating Droplets on a Printed Circuit Board,” filed on filed onJan. 30, 2006; U.S. Pat. No. 6,773,566, entitled “ElectrostaticActuators for Microfluidics and Methods for Using Same,” issued on Aug.10, 2004 and U.S. Pat. No. 6,565,727, entitled “Actuators forMicrofluidics Without Moving Parts,” issued on Jan. 24, 2000, both toShenderov et al.; Pollack et al., International Patent Application No.PCT/US2006/047486, entitled “Droplet-Based Biochemistry,” filed on Dec.11, 2006, the disclosures of which are incorporated herein by reference.Methods of the invention may be executed using droplet actuator systems,e.g., as described in International Patent Application No.PCT/US2007/009379, entitled “Droplet manipulation systems,” filed on May9, 2007. In various embodiments, the manipulation of droplets by adroplet actuator may be electrode mediated, e.g., electrowettingmediated or dielectrophoresis mediated. Examples of other methods ofcontrolling fluid flow that may be used in the droplet actuators of theinvention include devices that induce hydrodynamic fluidic pressure,such as those that operate on the basis of mechanical principles (e.g.external syringe pumps, pneumatic membrane pumps, vibrating membranepumps, vacuum devices, centrifugal forces, and capillary action);electrical or magnetic principles (e.g. electroosmotic flow,electrokinetic pumps piezoelectric/ultrasonic pumps, ferrofluidic plugs,electrohydrodynamic pumps, and magnetohydrodynamic pumps); thermodynamicprinciples (e.g. gas bubble generation/phase-change-induced volumeexpansion); other kinds of surface-wetting principles (e.g.electrowetting, and optoelectrowetting, as well as chemically,thermally, and radioactively induced surface-tension gradient); gravity;surface tension (e.g., capillary action); electrostatic forces (e.g.,electroosmotic flow); centrifugal flow (substrate disposed on a compactdisc and rotated); magnetic forces (e.g., oscillating ions causes flow);magnetohydrodynamic forces; and vacuum or pressure differential. Incertain embodiments, combinations of two or more of the foregoingtechniques may be employed in droplet actuators of the invention.

“Droplet operation” means any manipulation of a droplet on a dropletactuator. A droplet operation may, for example, include: loading adroplet into the droplet actuator; dispensing one or more droplets froma source droplet; splitting, separating or dividing a droplet into twoor more droplets; transporting a droplet from one location to another inany direction; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; cooling a droplet;disposing of a droplet; transporting a droplet out of a dropletactuator; other droplet operations described herein; and/or anycombination of the foregoing. The terms “merge,” “merging,” “combine,”“combining” and the like are used to describe the creation of onedroplet from two or more droplets. It should be understood that whensuch a term is used in reference to two or more droplets, anycombination of droplet operations that are sufficient to result in thecombination of the two or more droplets into one droplet may be used.For example, “merging droplet A with droplet B,” can be achieved bytransporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to volume of the resulting droplets(i.e., the volume of the resulting droplets can be the same ordifferent) or number of resulting droplets (the number of resultingdroplets may be 2, 3, 4, 5 or more). The term “mixing” refers to dropletoperations which result in more homogenous distribution of one or morecomponents within a droplet. Examples of “loading” droplet operationsinclude microdialysis loading, pressure assisted loading, roboticloading, passive loading, and pipette loading. Droplet operations may beelectrode-mediated. In some cases, droplet operations are furtherfacilitated by the use of hydrophilic and/or hydrophobic regions onsurfaces and/or by physical obstacles. Droplet operations may bediscrete flow operations, in which each overall operation involvesdiscrete steps, and each discrete step is mediated by the one or moreelectrodes upon which the droplets reside and/or adjacent electrodes. Incertain cases, discrete flow droplet operations may involve movement ofdroplets through a surrounding filler fluid, as compared to movement offiller fluid to cause droplet movements.

“Filler fluid” means a fluid associated with a droplet operationssubstrate of a droplet actuator, which fluid is sufficiently immisciblewith a droplet phase to render the droplet phase subject toelectrode-mediated droplet operations. The filler fluid may, forexample, be a low-viscosity oil, such as silicone oil. Other examples offiller fluids are provided in International Patent Application No.PCT/US2006/047486, entitled, “Droplet-Based Biochemistry,” filed on Dec.11, 2006; and in International Patent Application No. PCT/US2008/072604,entitled “Use of additives for enhancing droplet actuation,” filed onAug. 8, 2008.

“Immobilize” with respect to magnetically responsive beads, means thatthe beads are substantially restrained in position in a droplet or infiller fluid on a droplet actuator. For example, in one embodiment,immobilized beads are sufficiently restrained in position to permitexecution of a splitting operation on a droplet, yielding one dropletwith substantially all of the beads and one droplet substantiallylacking in the beads.

“Magnetically responsive” means responsive to a magnetic field.“Magnetically responsive beads” include or are composed of magneticallyresponsive materials. Examples of magnetically responsive materialsinclude paramagnetic materials, ferromagnetic materials, ferrimagneticmaterials, and metamagnetic materials. Examples of suitable paramagneticmaterials include iron, nickel, and cobalt, as well as metal oxides,such as Fe₃O₄, BaFe₁₂O₁₉, CoO, NiO, Mn₂O₃, Cr₂O₃, and CoMnP.

“Washing” with respect to washing a magnetically responsive bead meansreducing the amount and/or concentration of one or more substances incontact with the magnetically responsive bead or exposed to themagnetically responsive bead from a droplet in contact with themagnetically responsive bead. The reduction in the amount and/orconcentration of the substance may be partial, substantially complete,or even complete. The substance may be any of a wide variety ofsubstances; examples include target substances for further analysis, andunwanted substances, such as components of a sample, contaminants,and/or excess reagent. In some embodiments, a washing operation beginswith a starting droplet in contact with a magnetically responsive bead,where the droplet includes an initial amount and initial concentrationof a substance. The washing operation may proceed using a variety ofdroplet operations. The washing operation may yield a droplet includingthe magnetically responsive bead, where the droplet has a total amountand/or concentration of the substance which is less than the initialamount and/or concentration of the substance. Other embodiments aredescribed elsewhere herein, and still others will be immediatelyapparent in view of the present disclosure.

The terms “top” and “bottom” are used throughout the description withreference to the top and bottom substrates of the droplet actuator forconvenience only, since the droplet actuator is functional regardless ofits position in space.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletactuator, it should be understood that the droplet is arranged on thedroplet actuator in a manner which facilitates using the dropletactuator to conduct one or more droplet operations on the droplet, thedroplet is arranged on the droplet actuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet actuator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a cell sorting process conducted in a dropletactuator;

FIG. 2 illustrates a process of sorting droplets in a droplet actuatorby the types of cells contained therein;

FIGS. 3A and 3B illustrate side views of a first and second step,respectively, of a method of using a droplet actuator for separatingdifferent types of cells;

FIG. 4 illustrates a process for merging a droplet containing one ormore cells with a droplet of, for example, a reagent;

FIG. 5 illustrates a cell incubation process for growing cells in adroplet actuator, e.g., growing cells from a single cell;

FIG. 6 illustrates a cell fusing process of merging droplets thatcontain different types of cells;

FIG. 7 illustrates a process of separating different cell types by useof beads in a droplet actuator;

FIG. 8 illustrates a cell incubation process of growing cells on beadsin a droplet actuator; and

FIG. 9 illustrates a liquid exchange process in a cell culturereservoir.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of manipulating cells within a dropletactuator. For example, by use of operations, such as, dispensingdroplets from a cell suspension, analyzing the number of droplets in thedispensed droplet, merging the droplet with other droplets containingeither specific reagents or other cells, detecting a property of thedroplet, and incubating the droplet at a particular temperature.Embodiments of the invention provide a wide variety of techniques, ofwhich the following are examples: (1) sorting droplets by the number ofcells in a droplet, (2) sorting droplets by the types of cells in adroplet, (3) merging cell-containing droplets with reagent droplets,(4), incubating cell-containing droplets in order to grow more cells,(5) fusing droplets with different types of cells in a single droplet,(6) separating a single droplet with different types of cells intomultiple droplets, each with a reduced number of cell types, (7) growingcells on beads via incubation, (8) culturing cells in a culturereservoir, and (9) performing liquid exchange in a cell-containingculture reservoir.

8.1 Sorting Cell-Containing Droplets

FIG. 1 illustrates a cell sorting process 100 conducted in a dropletactuator. Droplets are dispensed from a parent droplet or reservoircontaining a suspension of cells and dispensed droplets are sorted bythe number of cells contained therein. FIG. 1 shows an arrangement ofelectrodes 110, e.g., electrowetting electrodes, in the dropletactuator. A sensor 114 is provided for detecting the number of cells ina droplet. Sensor 114 may be any suitable detection mechanism fordetecting the number of cells in a droplet. Examples include opticaldetection mechanisms, electrical detection mechanisms, andflorescence-based detection mechanisms. Cells may be labeled tofacilitate detection. A sample reservoir contains a volume of sampleliquid 118 that contains a quantity of cells 122. Droplet operations areused to dispense and transport droplets from the sample, such as adroplet 126. Each dispensed droplet may include a random number ofcells. Dispensed droplets are transported along electrodes 110 and intosensing proximity with sensor 114.

In one example scenario, the droplets of interest are those dropletsthat contain a single cell 122 only and any droplets that contain nocells 122 or two or more cells 122 are discarded or returned to thesample. In this example, when a droplet arrives at sensor 114, thenumber of cells 122 that are contained therein is determined. In thisexample, when single-cell droplets, such as single-cell droplets 130,are detected, single-cell droplets 130 are transported along a certainelectrode path for further processing. In contrast, when droplets thatcontain no cells 122 or two or more cells 122, such as droplets 134, aredetected, droplets 134 are transported along a certain differentelectrode path that returns droplets 134 back to the source volume ofsample liquid 118 or, alternatively, to a waste reservoir (not shown).The parent droplet may have a concentration of cells selected tostatistically (e.g., using Poisson distribution statistics) maximize thenumber of dispensed droplets including single cells. Droplet operationsmay be used to dilute excessively concentrated parent droplets in orderto improve or maximize the occurrence of dispensed droplets with singlecells.

Cell sorting process 100 of sorting droplets by the number of cells isnot limited to targeting and processing single-cell droplets only. Thetarget droplets of interest may contain any desired number of cellsdepending on the intended purpose of the droplet/cell operations withinthe droplet actuator. For example, two-celled droplets may be targetedand all others are discarded, one- or two-celled droplets may betargeted and all others are discarded, and so on.

FIG. 2 illustrates a process 200 of sorting droplets in a dropletactuator by the types of cells contained therein. FIG. 2 shows anarrangement of electrodes 210, e.g., electrowetting electrodes, whereinthe location of a sensor 214 is arranged along a transport path fordetecting the cell type in a droplet. Sensor 214 may be any suitabledetection mechanism for detecting the cell type in a droplet, such as,but not limited to, optical detection mechanisms, electrical detectionmechanisms, and florescent-based detection mechanisms. A samplereservoir contains a volume of sample liquid 218 that contains aquantity of various types of cells. In one example, sample liquid 218contains a quantity of a first cell type 222 and a quantity of a secondcell type 224. Droplet operations are used to dispense droplets thatcontain a random number and cell type and transport the dispenseddroplets into proximity with sensor 214.

In one example scenario, the droplets of interest are those dropletsthat contain the first cell type 222 only and any droplets that containno cells at all or at least one of the second cell type 224 arediscarded. Therefore, when a droplet arrives at sensor 214, the type(s)of cells contained therein is determined. In this example, when dropletsthat contain one or more of the first cell type 222 only, such asdroplets 226, are detected, droplets 226 are transported along a certainelectrode path for forming a sample volume 230 that contains the firstcell type 222 only. By contrast, when droplets that contain no cells atall or at least one of the second cell type 224, such as droplets 234,are detected, droplets 234 are transported along a different electrodepath for forming a waste volume 238 that may contain both the first celltype 222 and the second cell type 224. Alternatively, an electrode path(not shown) may be provided for forming a sample volume that containsthe second cell type 224 only.

In another example, the sorting process is used to enrich theconcentration of one cell type relative to another cell type. Forexample, any droplet containing the target cell type may be sorted toone location while any droplet not containing the target cell type maybe sorted to a second location. Thus, the first location is enrichedwith the target cell type while the second location is depleted of thetarget cell type. This process can be repeated any number of times toachieve a desired level of purification. When the target cell type islabeled, for example, with a fluorescent tag, the sensor may simply needto detect whether or not any signal is present in the droplet to performthis process. For more concentrated cell suspensions the sensor may beused to detect whether the total signal of the droplet exceeds a certainthreshold indicating whether the droplet is enriched or depleted of thetarget cell type. The process can be repeated many times over so thateven a relatively small enrichment at each step can produce asubstantial amount of purification.

Cell sorting process 200 is not limited to processing two types of cellsonly. Any number of types of cells may be detected and sortedaccordingly into any number of cell type-specific sample volumes. By useof a cell sorting process, such as cell sorting process 200, theinvention provides a method of providing droplets with enriched or pureconcentrations of pre-selected cell types.

FIGS. 3A and 3B illustrate side views of a first and second step,respectively, of a method of using a droplet actuator 300 for separatingdifferent types of cells. Droplet actuator 300 includes a top plate 310and a bottom plate 314 that are arranged with a gap therebetween. A setof electrodes 318, e.g., electrowetting electrodes, are associated withbottom plate 314. A quantity of sample fluid 322 is provided in the gapof droplet actuator 300. Additionally, sample fluid 322 contains aquantity of cells 326 of interest that are intermixed with a quantity ofother types of cells 330. Furthermore, when the dielectric properties ofthe different types of cells within sample fluid 322 are different,certain electrodes 318 may be used to manipulate certain cells by use ofdielectrophoresis (DEP). DEP is the lateral motion imparted on unchargedparticles (e.g., cells) as a result of polarization that is induced bynon-uniform electric fields (e.g., induced via electrodes 318). Forexample, FIG. 3A shows a certain electrode 318 that is near one end ofthe slug of sample fluid 322 is energized in a manner that correspondsto the dielectric properties of the cells 326 of interest. In doing so,the cells 326 of interest are attracted and immobilized (due to DEP)near one end of the slug of sample fluid 322, as shown in FIG. 3A, whilethe other types of cells 330 that have different dielectric propertiesare not attracted.

FIG. 3B shows that once the cells 326 of interest are attracted andimmobilized (due to DEP) near one end of the slug of sample fluid 322, adroplet splitting operation may occur in order to create a droplet 334of sample fluid that contains substantially the cells 326 of interestonly. By use of the method shown in FIGS. 3A and 3B, cells of interestare separated from unwanted cells via splitting. In another embodiment,DEP may be used to enrich a droplet with cells of interest, and a cellsorting method such as the method described with respect to FIG. 2 maybe employed to further isolate a specific cell type.

In an alternative embodiment, different types of beads that havedifferent affinities for different types of cells may be provided withinsample fluid 322. In one example, certain beads within sample fluid 322may have an affinity for the cells 326 of interest and substantially noaffinity for the other types of cells 330 and, thus, the cells 326 ofinterest only bind to these certain beads. Additionally, the beads mayhave different magnetic properties, for example, by having magneticallyresponsive beads of different sizes, by providing a mix of magneticallyresponsive beads and non-magnetically responsive beads, and anycombination thereof As a result, a magnetic field strength thatcorresponds to the beads that have an affinity for the cells 326 ofinterest may be applied in order to attract and immobilize the targetbeads near one end of the slug of sample fluid 322. Again, a subsequentdroplet splitting operation may occur in order to create a droplet 334of sample fluid that is enriched for the cells 326 of interest orcontains substantially the cells 326 of interest only.

8.2 Merging Droplets Containing Cells

FIG. 4 illustrates a process 400 for merging a droplet containing one ormore cells with a droplet of, for example, a reagent. FIG. 4 shows anarrangement of electrodes 410, e.g., electrowetting electrodes, alongwhich a cell-containing droplet, such as a cell-containing droplet 414,and a droplet of reagent, such as reagent droplet 418, may bemanipulated. In particular, a first step of cell merging process 400shows cell-containing droplet 414 and reagent droplet 418 beingtransported toward one another along electrodes 410 via electrowetting.A second step of cell merging process 400 shows a merged droplet 422,which is cell-containing droplet 414 and reagent droplet 418 that havebeen combined into a single droplet. The reagent may, for example,include a nutrient or other reagent for which the cell has a metabolicrequirement, a drug or other molecule used to perform a treatment on thecell, such a lysis reagent, or any chemical useful for performing ananalysis on the cell.

8.3 Incubating Cells in Droplets

FIG. 5 illustrates a cell incubation process 500 for growing cells in adroplet actuator, e.g., growing cells from a single cell. FIG. 5 showsan arrangement of electrodes 510, e.g., electrowetting electrodes, alongwhich a cell-containing droplet, such as a cell-containing droplet 514may be manipulated. In particular, a first step of cell incubationprocess 500 shows cell-containing droplet 514 that contains, forexample, a single cell only. A second step of cell incubation process500 is a temperature control step that maintains cell-containing droplet514 at a temperature that promotes cell growth. The second step shows anincubated droplet 518, which is a droplet that contains multiple cellsthat have grown over time from the single cell. By use of a cellincubation process, such as cell incubation process 500, cells canproliferate within a droplet actuator. By use of a cell sorting processas described above with respect to FIGS. 1 and 2, and an incubationprocess, droplets may be obtained having a substantially pure populationof cell types.

8.4 Fusing Cells in Droplets

FIG. 6 illustrates a cell fusing process 600 of merging droplets thatcontain different types of cells. FIG. 6 shows an arrangement ofelectrodes 610, e.g., electroweffing electrodes, along which a dropletthat contains a first cell type, such as a droplet 614, and a dropletthat contains a second cell type, such as droplet 618, may bemanipulated. In particular, a first step of cell fusing process 600shows droplet 614 and droplet 618 being transported toward one anotheralong electrodes 610 via electrowetting. A second step of cell fusingprocess 600 shows a fused droplet 622, which is droplet 614 and droplet618 that have been combined into a single droplet that contains both thefirst and second types of cells, e.g., fusion of a B-cell are with amyeloma cell to produce an antibody-producing hybridoma. In antherexample, a fusing process, such as cell fusing process 600, may be usedin the in vitro fertilization (IVF) process, i.e., fusing a sperm cellwith an egg cell.

8.5 Using Beads for the Manipulation of Cells

FIG. 7 illustrates a process 700 of separating different cell types byuse of beads in a droplet actuator. FIG. 7 shows an arrangement ofelectrodes 710, e.g., electroweffing electrodes, along which a dropletthat contains, for example, a first and second cell type, such as acell-containing droplet 714, and a droplet that contains beads, such asbead-containing droplet 718, may be manipulated. In particular, thebeads of bead-containing droplet 718 may be, for example, magneticallyresponsive beads. Examples of suitable magnetically responsive beads aredescribed in U.S. Patent Publication No. 2005-0260686, entitled,“Multiplex flow assays preferably with magnetic particles as solidphase,” published on Nov. 24, 2005. Additionally, the beads ofbead-containing droplet 718 may have an affinity for a certain celltype. In one example, the beads of bead-containing droplet 718 may havean affinity for the first cell type only and substantially no affinityfor the second cell type.

A first step of cell separation process 700 shows cell-containingdroplet 714 and bead-containing droplet 718 being merged alongelectrodes 710 using electrode-mediated droplet operations. A secondstep of cell separation process 700 shows a merged droplet 722, which iscell-containing droplet 714 and bead-containing droplet 718 that havebeen combined into a single droplet that contains both the first andsecond cell type along with the beads. The second step of cellseparation process 700 also shows that the first cell type within mergeddroplet 722 bind to the beads because the beads have an affinity for thefirst cell type only. By contrast, cells of the second cell type do notbind to the beads and, thus, remain substantially suspended withinmerged droplet 722. A third step of cell separation process 700illustrates a droplet-based wash procedure using wash buffer droplet 724that is used to remove the unbound second cell type while the beads arerestrained in place. The result is a cell-containing droplet 726 thathas a substantially pure cell type. The droplet 730 of unbound cells maybe subjected to further droplet operations and/or other processing oranalysis.

8.6 Growing Cells

FIG. 8 illustrates a cell incubation process 800 of growing cells onbeads in a droplet actuator. FIG. 8 shows an arrangement of electrodes810, e.g., electrowetting electrodes, along which a droplet thatcontains a certain cell type, such as a cell-containing droplet 814, anda droplet that contains beads, such as bead-containing droplet 818, maybe manipulated. In particular, the beads of bead-containing droplet 818may be, for example, magnetically responsive beads. Additionally, thebeads of bead-containing droplet 818 may have an affinity for theparticular cell type within cell-containing droplet 814.

A first step of cell incubation process 800 shows cell-containingdroplet 814 and bead-containing droplet 818 being transported toward oneanother along electrodes 810 via electrowetting. A second step of cellincubation process 800 shows a merged droplet 822, which iscell-containing droplet 814 and bead-containing droplet 818 that havebeen combined into a single droplet that contains both the cells and thebeads. The second step of cell incubation process 800 also shows thatthe cells within merged droplet 822 bind to the beads because the beadshave an affinity for the particular cell type. A third step of cellincubation process 800 is a temperature control step that maintainsmerged droplet 822 at a temperature that promotes cell growth. The thirdstep of cell incubation process 800 shows an incubated droplet 826,which is a droplet that contains multiple cells that have grown overtime upon the surface of the beads. By use of a cell incubation process,such as cell incubation process 800, cells can proliferate within adroplet actuator. In particular, the beads provide a means for growingcells on surfaces other than the droplet actuator surface so that thecells can be subsequently manipulated in the droplet actuator.

Embodiments of the invention may be provided for culturing cells on adroplet actuator. A cell-containing droplet, such as a droplet thatcontains one or more cells and/or cell-types, may be transported usingdroplet operations into contact with a cell culture medium. The cellculture medium may be included in a cell culture reservoir or well. Whennecessary, the cell culture medium may be in contact with the atmosphereor with a sub-atmosphere on the droplet actuator. The droplet actuatormay include or be associated with a heating element configured to heatthe cell culture medium to an appropriate temperature for incubation.

FIG. 9 illustrates a liquid exchange process 900 in a cell culturereservoir. FIG. 9 shows an arrangement of electrodes 910, e.g.,electrowetting electrodes, which fluidically connect a fluid reservoir914 and a cell culture droplet 918. The arrangement is useful, forexample, for performing a liquid exchange process supplying reagents,such as reagents metabolically useful substances, to cell culturedroplet 918. Fluid reservoir 914 may contain, for example, a volume ofreagent fluid 922. Cell culture droplet 918 may contain, for example, avolume of cell culture medium 926 that contains a quantity of cells 930.Cells 930 may be immobilized within cell culture droplet 918. In oneexample, cells 930 may be bound to magnetically responsive beads thatare within cell culture droplet 918, whereby the magnetically responsivebeads may be magnetically immobilized. Similarly, non-magneticallyresponsive beads may be physically immobilized, e.g., using one or morephysical barriers as described in International Patent Application No.PCT/US08/74151, filed on Aug. 25, 2008, entitled “Bead Manipulations ona Droplet Actuator,” the entire disclosure of which is incorporatedherein by reference. Any mechanism for immobilizing or retaining cells930 within cell culture droplet 918 is suitable. Liquid may be exchangedusing droplet operations for merging nutrient-containing droplets intocontact with cell culture droplet 918. In some cases, droplet splittingoperations may also be used to remove droplets including reducedquantities of such nutrients from the cell culture droplet 918.

In one example, by use of droplet operations, droplets of reagent fluid922 may be dispensed from fluid reservoir 914 and transported alongelectrodes 910 and into cell culture droplet 918. By introducing reagentfluid 922 into cell culture medium 926 of cell culture droplet 918,reagent fluid 922 is exchanged with cell culture medium 926.Subsequently, one or more droplets 934, which are formed of a mixture ofreagent fluid 922 and cell culture medium 926, are transported away fromcell culture droplet 918; all the while, cells 930 are held immobilizedwithin cell culture droplet 918. In alternative embodiments, cells 930are not immobilized.

Example purposes of a liquid exchange process, such as liquid exchangeprocess 900, may include, but are not limited to, delivering in ametered fashion various substances, such as metabolically usefulsubstances, drugs or chemicals, to cell culture medium 926 of cellculture droplet 918, changing the PH concentration of cell culturemedium 926 of cell culture droplet 918, changing the concentration ofcells 930 within cell culture medium 926 of cell culture droplet 918,and any combinations thereof.

8.7 Inoculation of a Cell Culture Medium

The droplet actuator of the invention may include a cell culture mediumarranged in sufficient proximity to one or more droplet operationselectrodes to permit a droplet comprising a cell to be introduced to theculture medium. The culture medium itself may be composed on the dropletactuator by combining various droplets including medium components. Theculture medium may or may not be subject to droplet operations. Inaccordance with the invention, a culture medium may be provided on thedroplet actuator. A droplet including one or more cells may betransported via droplet operations into contact with the culture medium.The inoculated culture medium may be incubated on the droplet actuator.A droplet may be contacted with a viscous culture medium and removedfrom the culture medium in order to capture one or more cultured cells,e.g., using the techniques described in International Patent ApplicationNo. PCT/US08/74151, filed on Aug. 25, 2008, entitled “Bead Manipulationson a Droplet Actuator,” the entire disclosure of which is incorporatedherein by reference.

8.8 Testing Cells

Cells on a droplet actuator may be tested using a wide variety oftechniques. A cell may be produced on the droplet actuator and tested onthe droplet actuator. A cell may be supplied from an external source tothe droplet actuator for testing. A reporter assay may be conductedusing droplet operations on the droplet actuator to determine whether agene of interest is being expressed. A RT-PCR assay may be conductedusing droplet operations on the droplet actuator using materialextracted from the cells using a droplet-based extraction protocol todetermine the presence and quantity of mRNA for the gene of interest. Animmunoassay may be conducted using droplet operations on the dropletactuator to determine the presence and the amount of protein produced.An enzymatic assay may be conducted using droplet operations on thedroplet actuator to determine the activity of the protein. Two or moreof these assays or assay types may be conducted on a single dropletactuator.

The results of a combination of the foregoing assays would show therelationship between the expression of the gene, the amount of proteinproduct and the activity of the protein. Cells may be treated withpathogens, therapeutic agents or other test substances or conditions,and the foregoing assays may be conducted to elucidate the effect of thetest substance on the cell.

CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention. Thisspecification is divided into sections for the convenience of the readeronly. Headings should not be construed as limiting of the scope of theinvention. The definitions are intended as a part of the description ofthe invention. It will be understood that various details of the presentinvention may be changed without departing from the scope of the presentinvention. Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limitation, as the presentinvention is defined by the claims as set forth hereinafter.

1. A method of inoculating a culture medium, the method comprising: (a)providing a droplet comprising a single cell type on a droplet actuator;and (b) inoculating a culture medium with the droplet.
 2. The method ofclaim 1 wherein step 1(b) comprises conducting one or more dropletoperations to bring the droplet into contact with the culture medium. 3.The method of claim 1 wherein step 1(a) comprises: (a) providing adroplet actuator comprising a sample droplet loaded thereon, the sampledroplet comprising cells of multiple cell types; (b) dispensing asub-droplet from the sample droplet; (c) analyzing the sub-droplet todetermine whether the sub-droplet comprises a single cell type; and (d)repeating steps (b) and (c) until one or more droplets each comprising asingle cell type is identified.
 4. A method of providing a dropletcomprising a single cell type, the method comprising: (a) providing adroplet actuator comprising: (i) a sample droplet loaded thereon, thesample droplet comprising cells of multiple cell types; and (ii) a beaddroplet comprising one or more beads having affinity for a specific oneof the cell types; (b) conducting one or more droplet operations tocombine the bead droplet with the sample droplet, thereby permittingcells of the specific one of the cell types to bind to the beads; and(c) conducting a droplet based washing protocol to separate the beadsbound to cells of the specific one of the cell types from cells of othercell types.
 5. The method of claim 1 wherein the droplet comprising asingle cell type is provided between droplet actuator substrates inproximity to a droplet operations surface.
 6. The method of claim 1wherein the droplet comprising a single cell type is providedsubstantially surrounded by a filler fluid.
 7. A method of providing adroplet having a modified distribution of cell types, the methodcomprising: (a) providing a droplet actuator comprising a sample dropletloaded thereon, the sample droplet comprising a first distribution ofcells of multiple cell types; (b) activating a series of electrodes toelongate the droplet, thereby providing an elongated droplet; (c)applying a dielectrophoretic gradient along the elongated droplet; and(d) deactivating an intermediate one of the series of electrodes todivide the droplet into two or more sub-droplets, each such sub-droplethaving a distribution of cells that differs from the first distributionof cells of multiple cell types.
 8. The method of claim 7 wherein atleast one of the sub-droplets provided in step 7(d) comprises a celltype that is enriched relative to the sample droplet.
 9. The method ofclaim 7 wherein the cell culture droplet and the second droplet aresituated between droplet actuator substrates in proximity to a dropletoperations surface.
 10. The method of claim 7 wherein the cell culturedroplet is substantially surrounded by a filler fluid.
 11. A method ofproviding a metabolically useful substance to a cell culture, the methodcomprising: (a) providing a droplet actuator comprising: (i) a cellculture droplet loaded thereon, the sample droplet comprising cells anda cell culture medium; and (ii) a second droplet comprising ametabolically useful substance; and (b) conducting one or more dropletoperations to combine the cell culture droplet with the second dropleton the droplet actuator.
 12. The method of claim 11 wherein the cellculture droplet is situated between droplet actuator substrates inproximity to a droplet operations surface.
 13. The method of claim 11wherein the cell culture droplet and the second droplet are situatedbetween droplet actuator substrates in proximity to a droplet operationssurface.
 14. The method of claim 11 wherein the cell culture droplet issubstantially surrounded by a filler fluid.
 15. The method of claim 11wherein the cell culture droplet and the second droplet aresubstantially surrounded by a filler fluid.
 16. A method of growingcells on a droplet actuator, the method comprising: (a) providing adroplet actuator comprising a cell culture droplet comprising a cellculture medium and one or more cells; and (b) maintaining the droplet ata temperature suitable for causing the cells to grow in the cell culturemedium on the droplet actuator.
 17. The method of claim 16 wherein thecell culture droplet is situated between droplet actuator substrates inproximity to a droplet operations surface.
 18. The method of claim 16wherein the cell culture droplet is surrounded by a filler fluid. 19.The method of claim 16 wherein the cells comprise cells bound to beads.20. A method of providing a hybridoma, the method comprising: (a)providing a droplet actuator comprising: (i) a B-cell droplet comprisinga B-cell; and (ii) a myeloma cell droplet comprising a myeloma cell; and(b) conducting droplet operations to combine the B-cell droplet with themyeloma cell droplet under conditions suitable to cause the fusion ofthe B-cell with the myeloma cell to produce a hybridoma.